Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09

Abstract Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C−H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co‐synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non‐heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

sMMO production and purification E. coli BL21 with the plasmids pLL319 + pBB528 + pBB541 for the co-production of the chaperonin GroES/EL and additional pZD04 (MmoZ) were grown in rich Terrific Broth (TB) medium at 37 °C until OD600nm of 2 and were then induced with 2 mM toluate and 1 mM IPTG. The protein production phase was performed at 18 °C for 30 h. The harvested cells were resuspended in twice their volume of resuspension buffer (500 mM NaCl, 50 mM KPO4, pH 7.2 containing additional Protease Inhibitor (EDTA-free, Roche) and DNase I). After two passages through a chilled French press at a pressure of 6.2 MPa, the suspension was centrifuged at 100,000x g for 45 min. The soluble extract was applied to a 2 mL Strep-tag Superflow affinity chromatography column, washed with 6 mL of resuspension buffer with protease inhibitor and eluted with 12 mL of 5 mM desthiobiotin solution. The eluate was then concentrated in an Amicon Ultra-15 centrifugal cell (100K membrane; Amicon, Witten, Germany). To remove desthiobiotin after Strep-tag Superflow affinity chromatography, the elution buffer of MMOH was exchanged via an illustra NAP-10 column (GE Healthcare UK Limited, Buckinghamshire, UK) to 25 mM MOPS, 100 mM NaCl, 0.2 mM (NH4)2Fe(SO4)2, 1 mM TCEP, pH 7.2. Protein concentration was determined with BCA protein assay kit (Pierce, USA) as described previously. [5] We isolated about 0.83 ± 0.63 mg (average of total five purifications) homogenous MMOH from 1 g cell pellet (wet weight). In order to monitor the purification, samples of every purification step were analysed by SDS-PAGE ( Figure S13). In the elution fraction, the pronounced bands were detected, which correspond to the three MMOH subunits calculated size of Strep-tagged-MmoX 62.0 kDa, MmoY 45.1 kDa, and MmoZ 18.9 kDa. A weak second band above the MmoZ band indicates the 6xHis-tagged MmoZ derivative (calculated size 20.5 kDa). Because of the already high purity of MMOH (> 95%), we decided to not proceed with further purification steps.

Spectroscopic measurements
Samples UV/visible spectra were recorded with a Varian Cary 60 (Agilent Technologies) at 20°C. The final working concentration of protein samples was around 45 mg/ml. Measurements were performed in 25 mM MOPS buffer with 100 mM NaCl at pH 7.2.
Electron Paramagnetic Resonance (EPR) spectroscopy was conducted on a Brucker EMX plus X-Band spectrometer equipped with an ER 4122 super-high Q resonator and an Oxford ESR900 helium flow cryostat. An Oxford ITC4 temperature controller was used for adjusting the temperature. The baseline was corrected by subtracting a reference spectrum of buffer solution recorded with the same experimental parameters. For subsequent corrections a spline function was used. Experimental parameters used were: microwave power 1 mW, microwave frequency 9.29 GHz, modulation amplitude 10 G, and modulation frequency 100 kHz. The as isolated MMOH was analyzed at a concentration of 55 µM and a volume of 100 µL.

Optical emission spectroscopy
For the determination of iron in MMOH, metal analysis was performed using a Perkin-Elmer Optima 2100DV inductively coupled plasma-optical emission spectrometer (Perkin-Elmer, Fremont, CA, USA) following the protocol described previously [6] . In short, 500 µL of protein samples were incubated overnight with equal amount of 65% nitric acid (Suprapur, Merck KGaA, Darmstadt, Germany) at 100 °C. Samples were filled up to 5 mL with water prior to ICP-OES analysis. Buffer samples without protein were treated the same way to check if footprint of metal is dissolved in the buffer. As reference, the multielement standard solution XVI (Merck) was used.

Activity measurements
The hydroxylation activity of MMOH was measured with nitrobenzene as the substrate under aerobic conditions (75 mM NaCl, 25 mM MOPS buffer, pH 8.0, 1 mM nitrobenzene). Proteins MMOH, MmoB and MmoC were added in the ratio H:B:C = 2:2:1. The reaction was started by adding 0.5 mM NADH. The production of p-nitrophenol was followed at 420 nm (VARIAN Cary 50 BIO UV-Visible Spectrometer, ε = 11.861 mM -1 cm -1 at pH 8.0). The production of pnitrophenol is followed at 405 nm due to interferences with NADH at shorter wavelength. [7] For the determination of reaction optima, first the optimum for salt concentration, and then the pH optimum were determined at 37 °C. Finally, the determination of the temperature optimum was performed. Salt concentrations were analysed in the range of 0-0.5 M NaCl (due to marine habitat from M. methylomonas), pH values ranged from 6.5-8.5, and temperature studies were performed from 10-42 °C. Furthermore, activity assays with addition of different catalase concentrations (0-8550 U/mg) were performed, because of the possibility of H2O2 production by MmoC, which could inhibit the sMMO activity. For the determination of optima conditions, no catalase was added. Each measurement was performed with technical triplicates but also with biological replicates.